This application is based on Patent Application Nos. 11-107258 (1999) filed on Apr. 14, 1999 in Japan and 2000-107103 (2000) filed on Apr. 7, 2000, the content of which is incorporated hereunto by reference.
1. Field of the Invention
The present invention relates to separation and assay of lipids in a sample such as serum or plasma in the field of medical research, biological research and the like. More specifically, the present invention relates to a method for separating and assaying lipoproteins contained in serum or plasma by means of electrophoresis; an assembly for performing such a method; and a system including such an assembly for separating and assaying lipoproteins, using electrophoresis to estimate the modific ation of lipoprotein by detecting quantitative and qualitative abnormalities of apoprotein in the lipoprotein.
2. Description of the Related Art
Lipoprotein in serum or plasma is a complex of lipid and protein, which includes cholesterol, phospholipid, and neutral fat. Therefore, an abnormality of lipoprotein reflects an abnormality of lipid metabolism, which can be estimated by a process including the steps of separating lipoproteins contained in serum or plasma and performing quantitative and qualitative analyses of the lipoprotein fraction with respect to any abnormalities thereof. Such a method for separating and assaying lipoprotein is one of the important clinical tests for the diagnosis and treatment of a disease affecting lipid metabolism such as hyperlipemia, coronary arteriosclerosis, hypothyroidism, obstructive liver disease, diabetes mellitus, and renal insufficiency.
As an approach to fractionating lipoproteins in a sample, there have been known methods using ultracentrifugal and electrophoretic techniques. The electrophoresis method uses an agarose film, a cellulose-acetate film, or a polyacrylamide gel(PAG) as a plate.
In the ultracentrifugal separation method, lipoproteins in serum can be separated from each other with respect to their respective specific gravities. The resulting fractions are generally referred as: High Density Lipoprotein (HDL); Low Density Lipoprotein(LDL); Very low Density Lipoprotein(VDL); and Chylomicron(CM), respectively.
Regarding the ultracentrifugal separation method, however, it takes much time to prepare a sample to be subjected to an ultracentrifugal machine and to fractionate such a sample. In addition, even finer fractionation of the sample is necessary for finer and detailed separation of lipoproteins to understand conditions during the separation. For this reason, it is difficult to perform the method of separating and assaying lipoprotein by means of ultracentrifugation in a clinical examination as a routine way to detect any abnormality in the target fraction or characterize the results of the separation for a specific disease.
In the electrophoretic separation method, on the other hand, lipoproteins in the solution can be fractionated mainly with respect to differences in their electrophoretic mobilities. In this case, the conditions of lipoprotein separation can be understood visually. For this reason, electrophoretic separation is often used in routine examination for separating and assaying lipoproteins in a sample. It is noted that the results of both electrophoretic and ultracentrigual procedures for separating lipoproteins show good correlation with each other. When a lipoprotein is fractionated using the electrophoretic technique, lipoprotein is separated into the fractions of xcex1(HDL), pre xcex2(VLDL), xcex2(LDL), and the point of origin(cylomicron). These fractions correspond to lipid species visualized by a lipid stain such as fat red 7B for estimating the amount of lipid in each fraction. For the convenience of one of skill in the art, the name of each lipoprotein fraction obtained by the prior art ultracentrifugal separation is described in the parentheses attached to each fraction name of the above electrophoresis.
The fundamental structure of lipoprotein is composed of a core part formed of a neutral fat(triglyceride) and cholesterol ester, a single hydrophilic lipid membrane that covers the core part, and one or more apoproteins adhered to the surface of such a membrane. The type of the apoprotein differs for each fraction of lipoprotein. That is, mainly, apoproteins in HDL are apoprotein A-I, A-II, A-IV, C-I, C-II, C-III, and E. Apoproteins in VLDL are apoprotein C-I, C-II, E, and B-100. Apoprotein in LDL is apoprotein B-100. Apoproteins in chylomicron are apoprotein A-I, A-II, A-IV, C-I, C-II, C-III, E, and B-48. In the electrophoretic process, a difference in the isoelectric point of each apoprotein contained in the lipoprotein is reflected as a difference in mobility of each fraction.
Since arteriosclerosis is a cause of adult diseases such as coronary arteriosclerosis, it is an important problem in basic and clinical medicine to prevent or treat arteriosclerosis. In particular, because of the recent increase in the number of patients exhibiting arteriosclerosis at young ages, and because patients with diabetes are liable to develop arteriosclerosis, management of lipid in serum is required to be stricter. For example, from epidemiological investigations conducted by the Framingham Institute and the like, it has been shown that the serum cholesterol value, especially the cholesterol value of low-density lipoprotein(LDL), is considered to be a most important,factor in atherosclerosis, which is a cause of coronary artery diseases.
The basis of cholesterol in LDL causing arteriosclerosis is that a macrophage is foamed by taking in modified LDL through a particular receptor, and the remnant adheres onto the vascular wall. Therefore, a screening examination of the modified LDL is necessary.
Here, xe2x80x9cmodified LDLxe2x80x9d is a generic name of variously modified LDL such as oxidized LDL, acetyl LDL, saccharized LDL, and MDA-LDL(malondialdehyde LDL). Further, the term xe2x80x9cmodificationxe2x80x9d used in the present specification means modification in a broad sense as normally used by one of skill in the art for modification of lipid or protein. For example, for a lipoprotein component contained in a blood sample, it includes not only modification caused by changes in physical or chemical environment in vivo such as a lipid metabolic anomaly in the body but also modification caused by changes in physical or chemical environment in vitro after sampling, which will be obvious to one of ordinary skill in the art without describing practical examples.
In general, it is said that small-dense LDL is liable to be doxidized. Therefore, as a screening examination of modified LDL, several methods have been tried to detect the ratio of small-dense LDL in lipoprotein. For example, there is a method by the electrophoretic process using PAG having a predetermined pore size and a high molecular sieving effect of PAG, first the smallest-particle (the largest mobility) HDL is separated, and then LDL, VLDL, and chylomicron are separated in order. Specifically, in the lipoprotein fractionation by the PAG electrophoretic process, lipoprotein is fractionated according to the difference in sizes of the particles contained in the lipoproteins, rather than by the difference in isoelectric points of particles contained in the lipoproteins.
For detecting the ration of small-dense LDL using the PAG electrophoretic process, there is a method using the relative distance between each fraction with respect to the position of the VLDL fraction. In this method, electrophoresis using a polyacrylamide gel is carried out, where the electrophoretic migration distance from the central position of the VLDL fraction to the central position of the LDL fraction is defined as xe2x80x9cxxe2x80x9d, and the electrophoretic migration distance from the central position of the VLDL fraction to the central position of the HDL fraction is defined as xe2x80x9cyxe2x80x9d; a relative electophoretic migration distance(Rf value or Rm value) is determined by the ratio(x/y) of these distances. The obtained value is compared with a value obtained for a normal standard sample in which the LDL is not small-dense. Here, the normal value means a value obtained by the same method for lipoprotein in which the LDL is not small-dense.
As described above, an object of the prior art method for separating and assaying lipoprotein by the PAG electrophoretic process is to determine the ratio of small-dense LDL in lipoprotein, wherein a relative migration distance of the LDL fraction is determined with respect to both VLDL and HDL fractions after electrophoresis. The relative migration distance reflects the difference in particle sizes, but does not reflect qualitative anomalies or quantitative anomalies of specific apoproteins each having different types or different numbers of apoproteins. Especially, lipoprotein fractionation utilizing PAG electrophoresis is not preferred for making a comparison between the normal LDL fraction having non-modified apoprotein B-100 and the modified LDL fraction in which the apoprotein B-100 is subject to some modification. On the other hand, an apoprotein in lipoprotein is directly responsible for a lipid metabolism in the body, so that the detection of an abnormality of the apoprotein may be very useful from a clinical standpoint.
For solving the problems described above and for simply determining a degree of modification of lipoprotein that reflects a quantitative or qualitative abnormality of apoproteins in lipoprotein, especially of modified LDL, based on its different isoelectric point in comparison with a normal one, a first object of the present invention is to provide a method for separating and assaying lipoprotein in a sample such as serum or plasma of human or mammals.
A second object of the present invention is to provide an assembly for performing such a novel method.
A third object of the present invention is to provide a system that includes such an assembly and optionally a computing device for controlling the assembly.
In a first aspect of the invention, there is provided an assembly for separating and assaying lipoprotein to determine a degree of modification of a predetermined component in a specimen using an electrophoretic pattern of a standard sample containing a lipoprotein having the predetermined component and an electrophoretic pattern of the specimen containing a lipoprotein having component of a same kind as the predetermined component, comprising:
an electrophoretic pattern preparation means for performing electrophoresis of the standard sample and the specimen to fractionate lipoprotein of each sample, then staining respective predetermined component using a reagent for detecting the respective predetermined component by staining the respective predetermined component in the respective lipoprotein to prepare respective electrophoretic pattern with visualized the respective predetermined component;
a waveform diagram preparation means for optically scanning the respective electrophoretic pattern with visualized the respective predetermined component and converting the respective electrophoretic pattern into an optical density waveform to prepare respective waveform diagram of the respective predetermined component; and
a means for judging the degree of modification of the predetermined component in the specimen by mathematically processing the respective electrophoretic patterns; wherein the means for judging the degree of modification of the predetermined component comprises:
a first means for determining a distance xe2x80x9caxe2x80x9d from an application point of the standard sample to a central position of a fraction corresponding to the predetermined component in the standard sample from electrophoretic pattern of the predetermined component of the standard sample;
a second means for determining a distance xe2x80x9cbxe2x80x9d from an application point of the specimen to a central position of a fraction corresponding to the predetermined component in the specimen from electrophoretic pattern of the predetermined component of the specimen; and
a third means for comparing the distance xe2x80x9caxe2x80x9d with the distance xe2x80x9cbxe2x80x9d to determine a relative mobility xe2x80x9cz(=b/a)xe2x80x9d of the specimen to the standard sample, wherein the degree of modification of the predetermined component in the specimen being judged on the basis of the relative mobility xe2x80x9czxe2x80x9d.
Here, the degree of modification of the predetermined component may be obtained as the ratio of modification xe2x80x9cMxe2x80x9d by substituting the relative mobility xe2x80x9cz(=b/a)xe2x80x9d into an equation of:
M=b/axe2x88x921=(bxe2x88x92a)/axe2x80x83xe2x80x83(1).
The degree of modification of the predetermined component may be obtained as a modification frequency xe2x80x9cMxe2x80x9d by substituting the relative mobility xe2x80x9cz(=b/a)xe2x80x9d into an equation of:
Mxe2x80x2=k(b/axe2x88x921)=k(bxe2x88x92a)/axe2x80x83xe2x80x83(2)
wherein xe2x80x9ckxe2x80x9d is a constant (k greater than 0).
The standard sample may be a standard serum not containing modified lipoprotein and the specimen may be a serum.
The predetermined component may be a low-density lipoprotein.
The predetermined component may be a lipid.
The electrophoresis may be carried out by an electrophoretic apparatus using a plate comprising an agarose gel as a main ingredient.
In a second aspect of the present invention, there is provided an assembly for separating and assaying lipoprotein to determine a degree of modification of a predetermined component in a specimen using an electrophoretic pattern of a standard sample containing a lipoprotein having the predetermined component, an electrophoretic pattern of the specimen containing a lipoprotein having component of a same kind as the predetermined component, and an electrophoretic pattern of an indicator sample containing a marker component capable of being an indicator of the predetermined component, comprising:
an electrophoretic pattern preparation means for performing electrophoresis of the standard sample and the specimen and the indicator sample to fractionate each sample, then staining respective components using reagents for detecting the predetermined component in respective fractions of the standard sample and the specimen, and the marker component in fraction of the indicator sample by staining thereby preparing respective electrophoretic patterns with visualized the respective components;
a waveform diagram preparation means for optically scanning the visualized respective electrophoretic patterns and converting the respective electrophoretic pattern into an optical density waveform to prepare respective waveform diagram of the respective predetermined component and the marker component; and
a means for judging the degree of modification of the predetermined component in the specimen by mathematically processing the respective electrophoretic patterns; wherein
the means for judging the degree of modification of the predetermined component have first measuring means and second measuring means, for judging the degree of modification of the predetermined component in the specimen based on a relative mobility of the indicator sample determined by the two measuring means, wherein
the first measuring means comprising:
a first means for determining a distance xe2x80x9caxe2x80x9d from an application point of the standard sample to a central position of a fraction corresponding to the predetermined component in the standard sample, from the waveform diagram of the standard sample,
a second means for determining a distance xe2x80x9ccxe2x80x9d from an application point of the indicator sample to a central position of a fraction corresponding to the marker component in the indicator sample, and
a third means for comparing the distance xe2x80x9caxe2x80x9d with the distance xe2x80x9ccxe2x80x9d and determining a relative mobility xe2x80x9cz1(=c/a)xe2x80x9d of the indicator sample to the standard sample,
the second measuring means comprising:
a fourth means for determining from the waveform diagram of the specimen a distance xe2x80x9cbxe2x80x9d from an application point of the specimen to a central position of a fraction corresponding to the predetermined component in the specimen, from the waveform diagram of the indicator sample a distance xe2x80x9ccxe2x80x9d from an application point of the indicator sample to a central position of a fraction corresponding to the marker component in the indicator sample, and
a fifth means for determining a relative mobility xe2x80x9cz2(=b/a=bxc2x7z1/c)xe2x80x9d of the specimen from the relative mobility xe2x80x9cz1xe2x80x9d determined by the first measuring means and the distance xe2x80x9cbxe2x80x9d and the distance xe2x80x9ccxe2x80x9d determined by the second measuring means.
Here, the degree of modification of the predetermined component may be obtained as the ratio of modification xe2x80x9cMxe2x80x9d by substituting the relative mobility xe2x80x9cz2(=b/a=bxc2x7z1/c)xe2x80x9d into an equation of:
M=bxc2x7z1/cxe2x88x921xe2x80x83xe2x80x83(3)
The degree of modification of the predetermined component may be obtained as modification frequency xe2x80x9cMxe2x80x2xe2x80x9d by substituting the relative mobility xe2x80x9cz2(=b/a=bxc2x7z1/c)xe2x80x9d into an equation of:
Mxe2x80x2=k(bxc2x7z1/cxe2x88x921)xe2x80x83xe2x80x83(4)
wherein xe2x80x9ckxe2x80x9d is a constant (k greater than 0)).
The standard sample may be a standard serum not containing modified lipoprotein, the specimen may be a serum, and the indicator sample may be a serum stored by adding a stabilizer.
The standard sample may be a standard serum not containing modified lipoprotein, the specimen may be a serum, and the indicator sample may contain an alcohol dehydrogenation enzyme capable of being a marker. In the second measuring means, furthermore, the fourth means may be a means for simultaneously determining the distance xe2x80x9cbxe2x80x9d and the distance xe2x80x9ccxe2x80x9d by subjecting a mixture of the indicator sample and the specimen to electrophoresis.
The first predetermined component may be low-density lipoprotein.
The first predetermined component may be lipid.
The electrophoresis may be carried out by an electrophoretic apparatus using a plate comprising agarose gel as a main ingredient.
In a third aspect of the present invention, there is provided a method for separating and assaying lipoprotein to determine a degree of modification of a predetermined component in a specimen using an electrophoretic pattern of a standard sample containing a lipoprotein having the predetermined component and an electrophoretic pattern of the specimen containing a lipoprotein having component of a same kind as the predetermined component, comprising:
an electrophoretic pattern preparation step for performing electrophoresis of the standard sample and the specimen to fractionate lipoprotein of each sample, then staining respective predetermined component using a reagent for detecting the respective predetermined component by staining the respective predetermined component in the respective lipoprotein to prepare respective electrophoretic pattern with visualized the respective predetermined component;
a waveform diagram preparation step for optically scanning the respective electrophoretic pattern with the visualized respective predetermined component and converting the respective electrophoretic pattern into an optical density waveform to prepare respective waveform diagram of the respective predetermined component; and
a step for judging the degree of modification of the predetermined component in the specimen by mathematically processing the respective electrophoretic patterns; wherein the step for judging the degree of modification of the predetermined component comprising:
a first step for determining a distance xe2x80x9caxe2x80x9d from an application point of the standard sample to a central position of a fraction corresponding to the predetermined component in the standard sample from electrophoretic pattern of the predetermined component of the standard sample;
a second step for determining a distance xe2x80x9cbxe2x80x9d from an application point of the specimen to a central position of a fraction corresponding to the predetermined component in the specimen from electrophoretic pattern of the predetermined component of the specimen; and
a third step for comparing the distance xe2x80x9caxe2x80x9d with the distance xe2x80x9cbxe2x80x9d to determine a relative mobility xe2x80x9cz(=b/a)xe2x80x9d of the specimen to the standard sample, wherein
the degree of modification of the predetermined component in the specimen being judged on the basis of the relative mobility xe2x80x9czxe2x80x9d.
Here, the degree of modification of the predetermined component may be obtained as the ratio of modification xe2x80x9cMxe2x80x9d by substituting the relative mobility xe2x80x9cz(=b/a)xe2x80x9d into an equation of:
M=b/axe2x88x921=(bxe2x88x92a)/axe2x80x83xe2x80x83(1)
The degree of modification of the predetermined component may be obtained as a modification frequency xe2x80x9cMxe2x80x2xe2x80x9d by substituting the relative mobility xe2x80x9cz(=b/a)xe2x80x9d into an equation of:
Mxe2x80x2=k(b/axe2x88x921)=k(bxe2x88x92a)/axe2x80x83xe2x80x83(2)
wherein xe2x80x9ckxe2x80x9d is a constant (k greater than 0)).
The standard sample may be a standard serum not containing modified lipoprotein and the specimen may be a serum.
The predetermined component may be a low-density lipoprotein.
The predetermined component may be a lipid.
The electrophoresis may be carried out by an electrophoretic apparatus using a plate comprising an agarose gel as a main ingredient.
In a fourth aspect of the invention, there is provided a recording medium storing a programming for performing the above method for separating and assaying lipoprotein.
In a fifth aspect of the invention, there is provided a system for separating and assaying lipoprotein, comprising: a computer for performing programming stored in the above recording medium, and an assembly for separating and assaying lipoprotein operating according to an instruction based on the programming from the computer.
In a sixth aspect of the invention, there is provided a method for separating and assaying lipoprotein to determine a degree of modification of a predetermined component in a specimen using an electrophoretic pattern of a standard sample containing a lipoprotein having the predetermined component, an electrophoretic pattern of the specimen containing a lipoprotein having component of a same kind as the predetermined component, and an electrophoretic pattern of an indicator sample containing a marker component capable of being an indicator of the predetermined component, comprising:
an electrophoretic pattern preparation step for performing electrophoresis of the standard sample and the specimen and the indicator sample to fractionate each sample, then staining respective components using reagents for detecting the predetermined component in respective fractions of the standard sample and the specimen, and the marker component in fraction of the indicator sample by staining thereby preparing respective electrophoretic patterns with visualized the respective components;
a waveform diagram preparation step for optically scanning the visualized respective electrophoretic patterns and converting the respective electrophoretic pattern into an optical density waveform to prepare respective waveform diagram of the respective predetermined component and the marker component; and
a step for judging the degree of modification of the predetermined component in the specimen by mathematically processing the respective electrophoretic patterns; wherein
the step for judging the degree of modification of the predetermined component has a first measuring step and a second measuring step, for judging the degree of modification of the predetermined component in the specimen based on a relative mobility of the indicator sample determined by the two measuring steps, wherein
the first measuring step comprising:
a first step for determining a distance xe2x80x9caxe2x80x9d from an application point of the standard sample to a central position of a fraction corresponding to the predetermined component in the standard sample, from the waveform diagram of the standard sample,
a second step for determining a distance xe2x80x9ccxe2x80x9d from an application point of the indicator sample to a central position of a fraction corresponding to the marker component in the indicator sample, from the waveform diagram of the indicator sample, and
a third step for comparing the distance xe2x80x9caxe2x80x9d with the distance xe2x80x9ccxe2x80x9d and determining a relative mobility xe2x80x9cz1(=c/a)xe2x80x9d of the indicator sample to the standard sample,
the second measuring step comprising:
a fourth step for determining from the waveform diagram of the specimen a distance xe2x80x9cbxe2x80x9d from an application point of the specimen to a central position of a fraction corresponding to the predetermined component in the specimen,
a fifth step for determining from the waveform diagram of the indicator sample a distance xe2x80x9ccxe2x80x9d from an application point of the indicator sample to a central position of a fraction corresponding to the marker component in the indicator sample, and
a sixth step for determining a relative mobility xe2x80x9cz2(=b/a=bxc2x7z1/c)xe2x80x9d of the specimen from the relative mobility xe2x80x9cz1xe2x80x9d determined by the first measuring step and the distance xe2x80x9cbxe2x80x9d and the distance xe2x80x9ccxe2x80x9d determined by the second measuring step.
Here, the degree of modification of the predetermined component may be obtained as a ratio of modification xe2x80x9cMxe2x80x9d by substituting the relative mobility xe2x80x9cz2(=b/a=bxc2x7z1/c)xe2x80x9d into an equation of:
M=bxc2x7z1/cxe2x88x921xe2x80x83xe2x80x83(3)
The degree of modification of the predetermined component may be obtained as a modification frequency xe2x80x9cMxe2x80x2xe2x80x9d by substituting the relative mobility xe2x80x9cz2(=b/a=bxc2x7z1/c)xe2x80x9d into an equation of:
Mxe2x80x2=k(bxc2x7z1/cxe2x88x921)xe2x80x83xe2x80x83(4)
wherein xe2x80x9ckxe2x80x9d is a constant (k greater than 0))
The standard sample may be a standard serum not containing modified lipoprotein, the specimen may be a serum, and the indicator sample may be a serum stored by adding a stabilizer.
The standard sample may be a standard serum not containing modified lipoprotein, the specimen may be a serum, and the indicator sample may contain an alcohol dehydrogenation enzyme capable of being a marker. In the second measuring step, furthermore, the fourth step and the fifth step are simultaneously performed by subjecting a mixture of the indicator sample and the specimen to electrophoresis.
The first predetermined component may be a low-density lipoprotein.
The first predetermined component may be a lipid.
The electrophoresis may be carried out by an electrophoretic apparatus using a plate comprising agarose gel as a main ingredient.
In a seventh first aspect of the invention, there is provided a recording medium storing a programming for performing the above method for separating and assaying lipoprotein.
In a eighth aspect of the invention, there is provided a system for separating and assaying lipoprotein, comprising: a computer for performing programming stored in the above recording medium, and an assembly for separating and assaying lipoprotein operating according to an instruction based on the programming from the computer.
When an electrophoretic apparatus of agarose plate is used in the above method for separating and assaying lipoprotein and assembly to be used for performing such a method, thin film of agarose is used for the plate. Alternatively, a thin film comprising a mixture of agarose and agar can also be used (such a plate is hereinafter referred to as agarose gel). This agarose plate in the form of agarose or agarose gel is preferably prepared from a solution using in a concentration range of 0.4%(W/W) to 1.2%(W/W), more preferably 0.5%(WJW) to 0.8%(W/W) to the buffer solution. Further, the applied voltage during electrophoresis is preferably 200 to 500V when an agarose thin film of 120 mm in length, 130 mm in width and 500 xcexcm in film thickness is used. Still further, it is preferable that a plate comprising agarose gel contains bovine serum albumin (BSA) of about 0.01 to 0.5%(W/W).
Instead of using the above agarose gel, an electrophoretic apparatus using a plate based on cellulose acetate, agar and the like or a capillary electrophoretic apparatus can be used.
The above and other objects, effects, features and advantages of the present invention will become more apparent from the following description of the embodiments thereof taken in conjunction with the accompanying drawings.